Metal-Modified Montmorillonite as Plasmonic Microstructure for Direct Protein Detection.

Thanks to its negative surface charge and high swelling behavior, montmorillonite (MMT) has been widely used to design hybrid materials for applications in metal ion adsorption, drug delivery, or antibacterial substrates. The changes in photophysical and photochemical properties observed when fluorophores interact with MMT make these hybrid materials attractive for designing novel optical sensors. Sensor technology is making huge strides forward, achieving high sensitivity and selectivity, but the fabrication of the sensing platform is often time-consuming and requires expensive chemicals and facilities. Here, we synthesized metal-modified MMT particles suitable for the bio-sensing of self-fluorescent biomolecules. The fluorescent enhancement achieved by combining clay minerals and plasmonic effect was exploited to improve the sensitivity of the fluorescence-based detection mechanism. As proof of concept, we showed that the signal of fluorescein isothiocyanate can be harvested by a factor of 60 using silver-modified MMT, while bovine serum albumin was successfully detected at 1.9 µg/mL. Furthermore, we demonstrated the versatility of the proposed hybrid materials by exploiting their plasmonic properties to develop liquid label-free detection systems. Our results on the signal enhancement achieved using metal-modified MMT will allow the development of highly sensitive, easily fabricated, and cost-efficient fluorescent- and plasmonic-based detection methods for biomolecules.

Disseminated Scedosporium apiospermum infection in a Maremmano-Abruzzese sheepdog.

BACKGROUND: Few cases of scedosporiosis have been reported in animals, but the true prevalence is probably underestimated due to a lack of awareness. Scedosporiosis in dogs has often been associated with localized infection (i.e., nasal infection, eumycetoma, or keratomycosis) or, in rare cases, disseminated infections. CASE PRESENTATION: This case report describes the clinical and pathological features and the diagnostic process of a rare systemic and fatal fungal infection in a dog caused by Scedosporium apiospermum. A 10-month-old female Maremmano-Abruzzese sheepdog showing weakness, lethargy, lateral decubitus, miosis and muscular rigidity was presented. Rodenticide poisoning was clinically suspected for the differential diagnosis. However, postmortem examinations revealed the presence of a swollen and soft subcutaneous nodule located near the right inguinal breast, which was associated with massive enlargement of the inguinal lymph nodes and small disseminated, cream-colored nodules in the kidneys and mesentery. Multiple fungal pyogranulomas were observed upon histological examination. Fungal isolation from the kidneys, breast and inguinal lymph nodes was performed. The internal transcribed spacer (ITS) sequences from the fungal colony DNA were searched in BLAST in the NCBI GenBank for species identification. The sequences of the fungi isolated from the kidney and breast cultures showed 100% sequence identity with sequences from Scedosporium apiospermum. CONCLUSIONS: This report shows that Scedosporium apiospermum may act as a primary pathogen in young and apparently healthy dogs and represents an important pathogen that should be considered during the diagnostic process, particularly when a fungal infection is suspected.

Multiplexed Discrimination of Single Amino Acid Residues in Polypeptides in a Single SERS Hot Spot.

The SERS-based detection of protein sequences with single-residue sensitivity suffers from signal dominance of aromatic amino acid residues and backbones, impeding detection of non-aromatic amino acid residues. Herein, we trap a gold nanoparticle in a plasmonic nanohole to generate a single SERS hot spot for single-molecule detection of 2 similar polypeptides (vasopressin and oxytocin) and 10 distinct amino acids that constitute the 2 polypeptides. Significantly, both aromatic and non-aromatic amino acids are detected and discriminated at the single-molecule level either at individual amino acid molecules or within the polypeptide chains. Correlated with molecular dynamics simulations, our results suggest that the signal dominance due to large spatial occupancy of aromatic rings of the polypeptide sidechains on gold surfaces can be overcome by the high localization of the single hot spot. The superior spectral and spatial discriminative power of our approach can be applied to single-protein analysis, fingerprinting, and sequencing.

3D nanoporous antennas as a platform for high sensitivity IR plasmonic sensing.

Nanoporous gold can be exploited as plasmonic material for enhanced spectroscopy both in the visible and in the near-infrared spectral regions. In particular, the peculiar morphology of such a substrate leads to a higher field confinement with respect to conventional plasmonic materials. This property can be exploited to achieve extremely high sensitivity to the changes in environmental conditions, making it an interesting tool for the development of sensors and biosensors. Here, we compared the sensitivity of a plasmonic resonator made of nanoporous gold with a similar structure made of homogeneous gold. To assess the enhanced sensitivity the same stoichiometric quantity of dielectric material was deposited via Atomic Layer Deposition onto the two considered structures. Experimental results proved the higher sensitivity was achievable using nanoporous gold. In particular, such 3D nanoporous structures can be proposed as a promising sensing platform in the near-infrared with a sensitivity over 4.000 nm/RIU.

Lab-on-a-Fiber Wearable Multi-Sensor for Monitoring Wound Healing.

Chronic wounds are regarded as a silent epidemic, affecting 1-2% of the population and representing 2-4% of healthcare expenses. The current methods used to assess the wound healing process are based on the visual evaluation of physical parameters. This work aims to design a wearable non-invasive device capable of evaluating three parameters simultaneously: the pH and the levels of glucose and matrix metalloproteinase (MMP) present in the wound exudate. The device is composed of three independent polymer optical fibers functionalized with fluorescent-based sensing chemistries specific to the targeted analytes. Each fiber is characterized in terms of detection sensitivity and selectivity confirming their suitability for monitoring the targeted parameters in ranges relevant to the wound environment. The selectivity and robustness of the multi-sensing device are confirmed with analyses using complex solutions with different pH levels (5, 6, and 7), different concentrations of glucose (1.25, 2.5, and 5 mm), and MMP (1.25, 2.5, and 5 µg mL-1 ). Given the simple set-up, the affordability of the materials used and the possibility of detecting additional parameters relevant to wound healing, such multi-sensing fiber-based devices could pave the way for novel non-invasive wearable tools enabling the assessment of wound healing from the molecular perspective.

Thioflavin-modified molecularly imprinted hydrogel for fluorescent-based non-enzymatic glucose detection in wound exudate.

The concentration of glucose in the body's fluids is an important parameter that can indicate pathological conditions such as the progress of infected wounds. Several wearables and implantable detection approaches have been developed with high selectivity and sensitivity for glucose. However, all of them have drawbacks such as low stability, limited selectivity, and often require complex technology. In this work, we present a fluorescent-based cost-efficient imprinted hydrogel (MIH_GSH) capable of detecting glucose within 30 â€‹min. The imprinting approach allows us to improve the selectivity for glucose, overcoming the low specificity and limited binding efficiency at neutral pH of boronic acid-based detection mechanisms. The binding affinity determined for glucose-MIH_GSH was indeed 6-fold higher than the one determined for the non-imprinted hydrogel with a calculated imprinting factor of 1.7. The limit of detection of MIH_GSH for glucose in artificial wound exudate was calculated as 0.48 â€‹mM at pH 7.4 proving the suitability of the proposed approach to diagnose chronic wounds (ca. 1 â€‹mM). MIH_GSH was compared with a commercial colorimetric assay for the quantification of glucose in wound exudate specimens collected from hospitalized patients. The results obtained with the two methods were statistically similar confirming the robustness of our approach. Importantly, whereas with the colorimetric assay sample preparation was required to limit the interference of the sample background, the fluorescent signal of MIH_GSH was not affected even when used to measure glucose directly in bloody samples. The sensing mechanism here proposed can pave the way for the development of cost-efficient and wearable point-of-care tools capable of monitoring the glucose level in wound exudate enabling the quick assessment of chronic injuries.

Ultrafast Determination of Antimicrobial Resistant Staphylococcus aureus Specifically Captured by Functionalized Magnetic Nanoclusters.

Sepsis, the systemic response to infection, is a life-threatening situation for patients and leads to high mortality, especially when caused by antimicrobial resistant pathogens. Prompt diagnosis and identification of the pathogenic bacteria, including their antibiotic resistance, are highly desired to yield a timely decision for treatment. Here, we aim to develop a platform for rapid isolation and efficient identification of Staphylococcus aureus, the most frequently occurring pathogen in sepsis. A peptide (VPHNPGLISLQG, SA5-1), specifically binding to S. aureus, was conjugated to the PEGylated magnetic nanoclusters, successfully enabling the specific capture and enrichment of S. aureus from blood serum. Consequently, fast detection of the antimicrobial resistance of the collected S. aureus was achieved within 30 min using a novel luminescent probe. These magnetic nanoclusters manifest a promising diagnostic prospect to combat sepsis.

pH-responsive silica nanoparticles for the treatment of skin wound infections.

Chronic wounds are not only a burden for patients but also challenging for clinic treatment due to biofilm formation. Here, we utilized the phenomenon that chronic wounds possess an elevated local pH of 8.9 and developed pH-sensitive silica nanoparticles (SiNPs) to achieve a targeted drug release on alkaline wounds and optimized drug utility. Chlorhexidine (CHX), a disinfectant and antiseptic, was loaded into SiNPs as the model drug. The loaded CHX displayed a release 4 - 5 fold higher at pH 8.0 and 8.5 than at pH 6.5, 7.0 and 7.4. CHX-SiNPs furthermore exhibited a distinctive antibacterial activity at pH 8.0 and 8.5 against both Gram-negative and -positive bacterial pathogens, while no cytotoxicity was found according to cell viability analysis. The CHX-SiNPs were further formulated into alginate hydrogels to allow ease of use. The antibacterial efficacy of CHX-SiNPs was then studied with artificial wounds on ex vivo human skin. Treatment with CHX-SiNPs enabled nearly a 4-lg reduction of the viable bacterial cells, and the alginate formulated CHX-SiNPs led to almost a 3-lg reduction compared to the negative controls. The obtained results demonstrated that CHX-SiNPs are capable of efficient pH-triggered drug release, leading to high antibacterial efficacy. Moreover, CHX-SiNPs enlighten clinic potential towards the treatment of chronic wound infections. STATEMENT OF SIGNIFICANCE: A platform for controlled drug release at a relatively high pH value i.e., over 8, was established by tuning the physical structures of silica nanoparticles (SiNPs). Incorporation of chlorhexidine, an antimicrobial agent, into the fabricated SiNPs allowed a distinctive inhibition of bacterial growth at alkaline pHs, but not at acidic pHs. The efficacy of the SiNPs loaded with chlorhexidine in treating wound infections was further validated by utilizing ex vivo human skin samples. The presented work demonstrates clinic potential of employing alkaline pH as a non-invasive stimulus to achieve on-demand delivery of antimicrobials through SiNPs, showcasing a valuable approach to treating bacterial infections on chronic wounds.

Changes in Optical Properties upon Dye-Clay Interaction: Experimental Evaluation and Applications.

The development of hybrid materials with unique optical properties has been a challenge for the creation of high-performance composites. The improved photophysical and photochemical properties observed when fluorophores interact with clay minerals, as well as the accessibility and easy handling of such natural materials, make these nanocomposites attractive for designing novel optical hybrid materials. Here, we present a method of promoting this interaction by conjugating dyes with chitosan. The fluorescent properties of conjugated dye-montmorillonite (MMT) hybrids were similar to those of free dye-MMT hybrids. Moreover, we analyzed the relationship between the changes in optical properties of the dye interacting with clay and its structure and defined the physical and chemical mechanisms that take place upon dye-MMT interactions leading to the optical changes. Conjugation to chitosan additionally ensures stable adsorption on clay nanoplatelets due to the strong electrostatic interaction between chitosan and clay. This work thus provides a method to facilitate the design of solid-state hybrid nanomaterials relevant for potential applications in bioimaging, sensing and optical purposes.

Detecting COVID-19 from Breath: A Game Changer for a Big Challenge.

Coronavirus disease 2019 (COVID-19) is probably the most commonly heard word of the last 12 months. The outbreak of this virus (SARS-CoV-2) is strongly compromising worldwide healthcare systems, social behavior, and everyone's lives. The early diagnosis of COVID-19 and isolation of positive cases has proven to be fundamental in containing the spread of the infection. Even though the polymerase chain reaction (PCR) based methods remain the gold standard for SARS-CoV-2 detection, the urgent demand for rapid and wide-scale diagnosis precipitated the development of alternative diagnostic approaches. The millions of tests performed every day worldwide are still insufficient to achieve the desired goal, that of screening the population during daily life. Probably the most appealing approach to consistently monitor COVID-19 spread is the direct detection of SARS-CoV-2 from exhaled breath. For instance, the challenging incorporation of reliable, highly sensitive, and cost-efficient detection methods in masks could represent a breakthrough in the development of portable and noninvasive point-of-care diagnosis for COVID-19. In this perspective paper, we discuss the critical technical aspects related to the application of breath analysis in the diagnosis of viral infection. We believe that, if achieved, it could represent a game-changer in containing the pandemic spread.

Galvanic Replacement Reaction as a Route to Prepare Nanoporous Aluminum for UV Plasmonics.

There is a growing interest in extending plasmonics applications into the ultraviolet region of the electromagnetic spectrum. Noble metals are commonly used in plasmonic, but their intrinsic optical properties limit their use above 350 nm. Aluminum is probably the most suitable material for UV plasmonics, and in this work we fabricated substrates of nanoporous aluminum starting from an alloy of Al2Mg3. The porous metal is obtained by means of a galvanic replacement reaction. Such nanoporous metal can be exploited to achieve a plasmonic material suitable for enhanced UV Raman spectroscopy and fluorescence. Thanks to the large surface to volume ratio, this material represents a powerful platform for promoting interaction between plasmonic substrates and molecules in the UV.

Electrophoretic Deposition of WS2 Flakes on Nanoholes Arrays-Role of Used Suspension Medium.

Here we optimized the electrophoretic deposition process for the fabrication of WS2 plasmonic nanohole integrated structures. We showed how the conditions used for site-selective deposition influenced the properties of the deposited flakes. In particular, we investigated the effect of different suspension buffers used during the deposition both in the efficiency of the process and in the stability of WS2 flakes, which were deposited on an ordered arrays of plasmonic nanostructures. We observed that a proper buffer can significantly facilitate the deposition process, keeping the material stable with respect to oxidation and contamination. Moreover, the integrated plasmonic structures that can be prepared with this process can be applied to enhanced spectroscopies and for the preparation of 2D nanopores.

Site-selective functionalization of plasmonic nanopores for enhanced fluorescence emission rate and Förster resonance energy transfer.

In this work, we use a site-selective functionalization strategy to decorate plasmonic nanopores with fluorescent dyes. Using an easy and robust fabrication method, we manage to build plasmonic rings on top of dielectric nanotubes with different inner diameters. The modulation of the dimension of the nanopores allows us to tailor their field confinement and their Purcell factor in the visible spectral range. In order to investigate how the changes in geometry influence the fluorescence emission rate efficiency, thiol-conjugated dyes are anchored on the plasmonic ring, thus forming a functional nanopore. We study the lifetime of ATTO 520 and ATTO 590 attached in two different configurations: single dye and FRET pair. For the single dye configuration, we observe that the lifetime of both single dyes decreases as the size of the nanopore is reduced. The smallest nanopores yield an experimental Purcell factor of 6. For the FRET pair configuration, we measure two regimes. For large nanopore sizes, the FRET efficiency remains constant. Whereas for smaller sizes, the FRET efficiency increases from 30 up to 45% with a decrease of the nanopore size. These findings, which have been supported by numerical simulations, may open new perspectives towards energy transfer engineering in plasmonic nanopores with potential applications in photonics and biosensing, in particular in single-molecule detection and sequencing.

SERS discrimination of single DNA bases in single oligonucleotides by electro-plasmonic trapping.

Surface-enhanced Raman spectroscopy (SERS) sensing of DNA bases by plasmonic nanopores could pave a way to novel methods for DNA analyses and new generation single-molecule sequencing platforms. The SERS discrimination of single DNA bases depends critically on the time that a DNA strand resides within the plasmonic hot spot. In fact, DNA molecules flow through the nanopores so rapidly that the SERS signals collected are not sufficient for single-molecule analysis. Here, we report an approach to control the residence time of molecules in the hot spot by an electro-plasmonic trapping effect. By directly adsorbing molecules onto a gold nanoparticle and then trapping the single nanoparticle in a plasmonic nanohole up to several minutes, we demonstrate single-molecule SERS detection of all four DNA bases as well as discrimination of single nucleobases in a single oligonucleotide. Our method can be extended easily to label-free sensing of single-molecule amino acids and proteins.

Site-Selective Integration of MoS2 Flakes on Nanopores by Means of Electrophoretic Deposition.

Here, we propose an easy method for site-selective deposition of two-dimensional (2D) material flakes onto nanoholes by means of electrophoretic deposition. This method can be applied to both simple flat nanostructures and complex three-dimensional structures incorporating nanoholes. The deposition method is here used for the decoration of large ordered arrays of plasmonic structures with either a single or few layers of MoS2. In principle, the plasmonic field generated by the nanohole can significantly interact with the 2D layer leading to enhanced light-material interaction. This makes our platform an ideal system for hybrid 2D material/plasmonic investigations. The engineered deposition of 2D materials on plasmonic nanostructures is useful for several important applications such as enhanced light emission, strong coupling, hot-electron generation, and 2D material sensors.

Coumarin-based, switchable fluorescent substrates for enzymatic bacterial detection.

Enzymatically-switchable fluorescent substrates, such as the commercially available 4-methyl umbelliferones (4-MU) are used as standard indicators of enzymatic activity for the detection of various microorganisms and pathogens. However, a major disadvantage of 4-MU is its relatively high pKa leading to only partial dissociation of the fluorescent anion under the conditions where the enzymes are most effective (pH 6-6.5). Here we present a method for new, enzymatically-switchable, fluorescent substrates with improved photo-physico/chemical properties. The lead derivative, 4-AAU, shows excellent solubility in aqueous media (0.81 mg/mL) when compared to 4-MU (0.16 mg/mL), significantly improved quantum yield and wider dynamic range of its fluorescence properties. The corresponding bacterial substrate ß-4-AAUG showed superior selectivity in the detection of clinically relevant amounts of E. coli, Enterococcus and K. pneumonia (1 CFU). The fluorescence intensity of ß-4-AAUG was almost 5 times higher than that of the standard, the detection was possible in reasonably short time (∼ 2.5 h) and with excellent sensitivity.

Improving colloidal stability of silica nanoparticles when stored in responsive gel: application and toxicity study.

When silica nanoparticles (SiNP) are stored in aqueous solution, even for few hours, they have a tendency to form agglomerates and therefore adapt inhomogeneous structures. Here we present a very practical method to store SiNP in responsive hydrogel. We have confirmed that SiNP kept in the responsive hydrogel do not undergo through undesirable morphological changes and while in storage they maintain their excellent colloidal stability. The effect of SiNP hollowing (i.e. dissolution of the core of the particles that leaves empty cavity inside) was significantly inhibited in the hydrogel, which is a critical feature for any nano-medical applications (e.g. controlled drug release). To demonstrate the applicability of the hydrogel-storing concept within a biologically relevant context, in this work we have evaluated the toxicological effects of the responsive SiNP-gel formulation in a model in vitro (human cell line U87GM and hemocompatibility using red blood cells) and ex ovo (hen's egg test) experiments. Particles stored in the gel as well as the pure gel did not affect the hemocompatibility (hemolysis and erythrocyte aggregation) up to a concentration of 100 µg/mL. Furthermore, systemic injections into the blood circulation of the chick area vasculosa confirmed the biocompatibility in a more complex biological environment. All evaluated toxicological values (hemorrhage, thrombosis, vascular lysis, and lethality) were comparable with the negative control, and no differences in toxicological response could be observed between the SiNP stored in hydrogel and the control nanoparticles stored in the solution.

Hybrid plasmonic nanostructures based on controlled integration of MoS2 flakes on metallic nanoholes.

Here, we propose an easy and robust strategy for the versatile preparation of hybrid plasmonic nanopores by means of controlled deposition of single flakes of MoS2 directly on top of metallic holes. The device is realized on silicon nitride membranes and can be further refined by TEM or FIB milling to achieve the passing of molecules or nanometric particles through a pore. Importantly, we show that the plasmonic enhancement provided by the nanohole is strongly accumulated in the 2D nanopore, thus representing an ideal system for single-molecule sensing and sequencing in a flow-through configuration. Here, we also demonstrate that the prepared 2D material can be decorated with metallic nanoparticles that can couple their resonance with the nanopore resonance to further enhance the electromagnetic field confinement at the nanoscale level. This method can be applied to any gold nanopore with a high level of reproducibility and parallelization; hence, it can pave the way to the next generation of solid-state nanopores with plasmonic functionalities. Moreover, the controlled/ordered integration of 2D materials on plasmonic nanostructures opens a pathway towards new investigation of the following: enhanced light emission; strong coupling from plasmonic hybrid structures; hot electron generation; and sensors in general based on 2D materials.